Improved method for harvesting human Schwann cells from mature peripheral nerve and expansion in vitro.

The use of cellular prostheses containing large populations of Schwann cells (SC) has been proposed as a future therapeutic approach in the repair of neural tissue. We have sought to define an efficient protocol for the harvest and expansion of human SC from mature human peripheral nerve. We evaluated SC proliferation occurring within fresh explants and studied the relationship between certain parameters (cell yield, purity, and rate of SC proliferation) and the conditions of maintenance of nerve explants prior to dissociation. In addition, we studied SC proliferation after dissociation in a variety of conditions. We observed that SC within explants divide at a low rate during the first 3 weeks following explantation; this proliferation falls to near zero during the fourth week. The cell yield, SC purity, and proliferation rate following dissociation were all increased when nerve explants were exposed to heregulin/ forskolin for 2 weeks prior to dissociation. Electron microscopic analysis showed that heregulin/forskolin exerted trophic effects on SC within explants. Following dissociation, SC growth in heregulin/forskolin-containing medium was more rapid on laminin or collagen than on poly-L-lysine. These results provide new insights into human SC biology and suggest several procedural improvements for harvesting and expanding these cells. The new method we describe shortens our previous procedure by 4-6 weeks and provides a 30-50-fold increase in the number of SC obtained relative to the earlier procedure.